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Reference mutant choicesl

The mutants below were chosen based on promising student results in previous years, then tested by the teaching faculty.

Mutation X#Z

Primer (forward)

Silent mutation

New restriction site

Known effect

D24H

C GAC AAG GAT GGG CAC GGT ACC ATC ACC -ANS

Gly25

KpnI

Affinity decrease and cooperativity increase

E67K

GGC AAT GGT ACG ATT TAC TTT CCT AAA TTT CTT ACT ATG ATG GC

Gly63

RsaI

Small affinity increase, and perhaps slight cooperativity decrease

T79P

GA AAA ATG AAG GAC CCA GAC AGC GAA GAG GAA ATC C

Thr79

PpuMI

Small affinity decrease and perhaps slight cooperativity increase

M124S

GAT GAA GAA GTT GAT GAA AGT ATA CGG GAA GCA GAT ATC GAT GG

Arg126

AccI

Affinity decrease and cooperativity increase

Note that we also investigated D22H and D129P, but both of these turned out to destroy calcium response, and in low salt conditions to eliminate fluorescence itself. The limited choices above aren't meant to imply that you must narrowly investigate these residues or ones just like them.

T/R

Please list all amino acid residues with respect to calmodulin itself rather than the chimeric protein inverse pericam.

Team colour

Mutation X#Z

Primer (forward)

Silent mutation

New restriction site

Reference mutant choice

Teaching example

S101L

GG AAC GGC TAC ATC CTC GCT GCG CAG TTA CGT CAC G

Ala 103

FspI

N/A

Red

Orange

Yellow

Green

Blue

Pink

Purple

Platinum

W/F

Please list all amino acid residues with respect to calmodulin itself rather than the chimeric protein inverse pericam.